When tissue has been traumatized, for example by wounding or burning, the healing process is extremely complex but known to be mediated by numerous protein factors. These factors are essential to cell growth and differentiation during the replacement of destroyed tissue. Many factors have been identified as candidate protein factors for healing based on the ability of various tissue extracts (e.g., brain, pituitary, and hypothalamus) to stimulate mitosis in cell culture. A number of acronyms have been given to the factors in these extracts, such as "PDGF" (platelet-derived growth factor), "MDGF" (macrophage-derived growth factor), "EGF" (epidermal growth factor), "TAF" (tumor angiogenesis factor), "ECGF" (endothelial cell growth factor), "FGF" (fibroblast growth factor), "HDGF" (hypothalamus-derived growth factor), "RDGF" (retina-derived growth factor), "HGF" (heparin-binding growth factor). See, e.g., Hunt, T. K., (1984) J- Trauma, 24:S39-S49; Lobb et al., (1984) Biochemistry 23:6295-6299.
Basic fibroblast growth factor (bFGF) was initially isolated from bovine pituitary glands and was identified by its ability to promote fibroblast proliferation. It has since been shown that bFGF acts on a range of cell types. Thus, bFGF has many potential uses including, for example, endothelial cell culture, inducing blood vessel growth, aiding wound healing, etc.
bFGF promotes the proliferation of sensitive target cells, such as vascular endothelial cell and fibroblasts (Gospodarowicz et al., (1987) Endocr. Rev., 8:95-114; Baird et al., (1986) Recent Prog. Horm. Res., 42:143-205). bFGF is chemotactic for a variety of cell types (Moscatelli et al., (1986) Proc. Nat'l Acad. Sci. U.S.A., 83:2091-2095; Senior et al., (1986) Biochem. Biophys. Res. Commun., 141:67-72; Presta et al., (1986) Mol. Cell. Biol., 6:4060-4066), and induces the synthesis of collagenase and plasminogen activator in endothelial cells (Moscatelli et al., supra).
Exogenously supplied bFGF has effects on wound healing (Davidson et al., (1985) J. Cell Biol., 100:1219-1227), bone healing (Canolis et al., (1987) J. Clin. Invest., 79:52-58), vascular grafting (Griesler et al., (1986) Trans. Am. Soc. Artif. Intern. Organs, 32:346-349), lens regeneration (Yamada (1982) Cell Biology of the Eye, McDevitt D. S. ed., pp. 193-234, Academic Press, New York), and limb regeneration (Gospodarowicz and Mescher (1981) Advances in Neurology: Neurofibromatosis, Riccardi V. M. and Mulvihill J. J. eds., Vol. 29, p. 149, Raven Press, New York). Basic FGF is angiogenic "in vivo" (Gospodarowicz et al., (1979) Exp. Eye Res., 28:501-514) and has neurotrophic properties (Morrison et al., (1986) Proc. Nat'l Acad. Sci. U.S.A., 83:7537-7541).
Heparin chromatography has been used to purify native FGF which has been derived from either crude, cell culture-derived lysates or relatively impure commercial preparations. Klagsbrun, M. (1985) Australian Patent Publication AU-A39206/85 discloses application of heparin chromatography to remove undesired impurities from a crude solution of endothelial cell-growth factor (ECG). After the ECG is immobilized on the heparin it is eluted using a salt solution and recovered from the eluate.
Recombinant bFGF has been produced and purified, again, using heparin affinity HPLC. Masaharu et al., (1988) Biochem. Biophys. Res. Commun., 151:701-708. Masaharu et al. used site-directed mutagenesis to change four cysteine residues of the mature bFGF protein to serine residues attempting to stabilize the protein and reduce the heterogeneity of bFGF elution from heparin affinity HPLC, while still retaining biological activity in some of the modified proteins.
Heparin, as used in the known bFGF purification methods, might affect affinity, rate of uptake and pharmacokinetics of bFGF in vitro. If the purified FGF is intended for therapeutic use in vivo, there may be some concern that heparin used to purify FGF could leach into the final product. Because heparin is used clinically to inhibit blood coagulation, it is an undesirable contaminant in therapeutic preparations of bFGF. Furthermore, heparin-Sepharose beads are a soft-bead, agarose gel that, when scaled up for commercial production, collapse under the high pressure used in process scale chromatography. In addition, the heparin purification process yields a product which is only about 95% pure. (Similarly, use of a non-heparin, ion exchange chromatography system to purify basic FGF yielded a product of 95% purity. Banks, EPO publication 275,204.)
Heparin is a critical element in a "biaffinity" purification process as discussed in Shing, (1988) J. Biol. Chem., 263:9059-9062 (hereinafter "Shing"). (See also PCT application WO 89/08117, published 8 Sep. 1989, which is directed to the Shing bFGF purification process as published in 1988; and PCT application WO 89/08144, published 8 September 1989, which is further directed to the purification of urokinase using the Shing biaffinity purification process.) As illustrated by Shing's FIG. 1, the procedure uses the differential affinities of bFGF for heparin and copper ion. After applying a sample containing partially purified bFGF to a column having both heparin and copper ion ligands, the column is washed with 2M NaCl to remove the heparin-bound contaminants while the bFGF remains bound to the copper. When the column is rinsed with 0.6M NaCl the FGF again attaches to both the heparin and the copper. A subsequent wash with 0.6M NaCl and 10 mM imidazole removes the contaminants bound to copper, but FGF, because of its affinity for heparin, remains bound to the column. A gradient of 0.6M NaCl to 2M NaCl plus 10 mM imidazole removes the bFGF forms from the column. Although Shing uses copper to bind bFGF, heparin is, however, half of the column material and so the potential for heparin to leach into the isolated growth factor is still present. (The additional problems of developing a commercially viable scale up and achieving ideal purity are also encountered in the Shing process.)
EPO disclosure 287,470 to Barritault et al. (hereinafter "Barritault"; page number references are to the English translation) shows the purification of growth factors, such as bFGF, using an amino acid derivative column that mimics a heparin column. It uses, e.g., high salt (as in Barritault claim 11) and is an ion exchange type of column. The ligand is prepared by derivatizing --SO.sub.2 Cl groups in the presence of MOH, wherein M is a physiologically acceptable metal, with a chosen amino acid to form --SO.sub.3 M groups and then --SO.sub.2 R groups. (Barritault page 5.) The R group is formed when an amino acid or amino acid derivative is bound to the resin in the presence of the MOH base. (Id.) Although the column may have "biological properties [that] are analogous to those of heparin" (Barritault page 4), the resultant yields are low and the derivatization method is cumbersome.
A number of non-heparin column chromatography systems have been developed for purification of materials other than bFGF. One form of affinity chromatography which does not use heparin is shown in Hughes et al. U.S. Pat. No. 4,431,546 (hereinafter "the '546 patent"). The '546 patent uses ligands of the reactive dye type wherein separation of a biological substance from a mixture is done in the presence of metal ions. See '546 patent, col. 1, lines 42-55. Metal ions are added to the crude solution and the dye is used to bind the desired biological material thereby removing it from the solution so that it may later be eluted in pure form. The metal ions apparently improve binding of the biological material to the ligand. See, e.g., the '546 patent, col. 1, lines 59-65. An inherent problem exists when reactive dyes are used as a ligand for purifying biological material such as FGF destined for use in humans or mammals, given the potential that such dyes are carcinogenic or toxic.
Metal affinity chromatography has not been used to purify bFGF, although various other purification procedures use metal affinity chromatography. See, e.g., Ryden et al., (1978) J. Biol. Chem., 253:519-524; Chadha et al., (1979) J. Gen. Virol., 43:701-706; Kikuchi et al. (1981) Analyt. Biochem., 115:109-112; Porath, J. (1983), Archives Biochem. and Biophysics, 225:543-547; Coppenhaver (1985) ICRS Med. Sci., 13:811-812; Weselake et al., (1986) Analyt. Biochem., 155:193-197; Sulkowski, E., (1987) Protein Purification: Micro to Macro (Alan Liss, Inc.) pp. 149-162. Those of skill in the metal affinity chromatography field acknowledge that "it is extremely difficult a priori to state which proteins will and which will not exhibit an affinity for immobilized metal ions." Smith, et. al., U.S. Pat. No. 4,569,794 (hereinafter "the '794 patent").
The '794 patent attempts to make protein purification using metal affinity chromatography universally applicable. To achieve this, the is directed to a universal capture peptide which binds to metal ions. Recombinantly produced hybrid molecules of the capture peptide and the peptide of interest are caught. After purification of the hybrid, the capture peptide portion of the hybrid is cleaved from the peptide of interest. Cleavage, however, is not straightforward and may result in either damage to the peptide of interest or may fail to remove undesired residues. Additional purification is then required to separate the peptide of interest from the capture peptide and the uncleaved hybrid. Furthermore, the process is not readily adaptable to purification of nonrecombinant proteins. In sum, although metal ion affinity chromatography has been useful for purifying some proteins, heparin affinity chromatography has been the only sure way to purify bFGF.
An ideal bFGF purification process would provide a method which avoided the use of heparin, yielded a protein that was at least 98% free of contaminating proteins, and could be scaled up for commercial production.